The present invention relates to mammalian cell based expression systems and the expression and secretion of recombinant proteins by using secretory signal peptides. The present invention also relates to an expression cassette useful for the secretion of a heterologous gene from a mammalian cell, in particular a CHO cell. The present invention is also directed to methods of secreting a heterologous protein from mammalian cells such as CHO cells and methods for the production of secreted recombinant proteins in mammalian host cells.
Recombinant polypeptides for medical, research and veterinary applications such as antibodies are produced using a wide variety of genetically engineered organisms that include prokaryotic and eukaryotic cells. However, a lot of these proteins are glycoproteins requiring post-translational modifications. Thus, prokaryotic host cells such as bacterial cells are not suitable. For this reason other protein expression systems were developed using the cells of higher eukaryotes, e.g. insect cells or mammalian cells. Viral expression systems can produce recombinant proteins both in insects and mouse cell lines but suffer from several serious drawbacks, in particular that purification of recombinant proteins from virus infected systems is very problematic.
A major problem in biotechnology exists in the production and recovery of recombinant polypeptides that are not readily secreted such as intracellular proteins or protein subunits, from genetically engineered organisms. Often these intracellular proteins or protein subunits can be expressed at only moderate levels inside a cell and their purification must first include steps to lyse the cells, followed by several procedures to isolate the desired polypeptides from the other intracellular proteins.
One approach to solve these problems is to have recombinant proteins secreted into the periplasmic space or culture medium. Whenever possible, secretion is the preferred strategy since it permits easy and efficient purification from the extracellular medium. In addition, the secretory production of recombinant proteins has the advantage that proteolytic degradation may be avoided and that there is a better chance of correct protein folding. Successful protein secretion requires effective translocation of the protein across the endoplasmatic reticulum or plasma membrane. Proteins that are secreted from a cell through a cell membrane are generally produced within the cell in a precursor form, referred to as a “preprotein”. This “preprotein” form includes an additional peptide sequence at the amino-terminus which is required to target the nascent peptide chain to the endoplasmatic reticulum to enable its entry into the secretory pathway. This additional peptide sequence is referred to as a signal sequence.
However, it is known that secretion frequently does not function to the degree desired, for example because the native signal sequence of the recombinant protein often does not operate well in the host cell. To date, each expression system needs specific tailoring to meet the requirements for each protein product to ensure correct folding, activity and desired yield. Although quite a lot of signal sequences have been identified which might be useful for the secretion of particular recombinant proteins, there is still a need in the art for additional signal sequences that can promote efficient secretion of recombinant proteins, in particular immunoglobulins, in mammalian host cells.
Thus, the technical problem underlying the present invention is to provide a method to efficiently secrete non-secretion competent polypeptides, in particular antibody chains from a eukaryotic host cell such as a Chinese Hamster Ovary (CHO) cell.
The present invention solves this technical problem by providing an expression cassette for the secretion of a heterologous protein from a mammalian host cell, in particular a CHO cell comprising a promoter, functionally linked to a DNA sequence encoding a signal peptide which is linked in frame to a DNA sequence encoding a heterologous protein, wherein the DNA sequence encoding the signal peptide is selected from the group consisting from SEQ ID No. 2, SEQ No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No.10 or a DNA sequence encoding an amino acid sequence depicted in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 or SEQ ID No. 9.